The goals of this project are to delineate the roles of the polymerase activity and the 3' to 5' exonuclease activity of DNA polymerase I in maintaining the fidelity of DNA synthesis, and to elucidate the mechanisms of template-directed polymerization and proof-reading at the molecular level. The mechanism of proof-reading has been investigated by inhibitor studies. Nucleoside 5'-monophosphates have been shown to be competitive inhibitors of the 3' to 5' exonuclease activity of DNA polymerase I when DNA is the variable substrate, whereas the polymerase activity is not inhibited. The metal chelator 1,10-phenanthroline has been shown to competitively inhibit the polymerase activity of DNA polymerase I when DNA is the variable substrate, whereas the 3' to 5' exonuclease activity is not inhibited. These results suggest that the polymerase and 3' to 5' exonuclease activities of DNA polymerase I are catalyzed by separate active sites and that the primer terminus of the primer/ template, which is a substrate for both activities, can bind to either of the catalytic sites.